The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB) phosphorylate fructose-6-phosphate (F6P) to fructose-2,6-bisphosphate (F2,6BP), which is an allosteric activator of 6-phosphofructo-1-kinase, a rate-limiting enzyme in the glycolytic pathway. Although there are four PFKFB enzymes, PFKFB3 and PFKFB4 are of particular interest since these enzymes have been found to be activated in human cancers, to be increased by hypoxic exposure via HIF-1?, and, in the case of PFKFB3, to be required for the growth of Ras-transformed tumors. In order to better understand the relative contributions of PFKFB2-4 to glycolysis, we examined the subcellular localization of these enzymes and were surprised to find that whereas PFKFB2 and PFKFB4 localized to the cytoplasm (the site of glycolysis), PFKFB3 localized to the nucleus. We then over-expressed PFKFB3 in HeLa cells and observed no change in glucose uptake but rather an increase in proliferation. Eukaryotic cell division is controlled by cyclin dependent kinases (CDKs) that bind to regulatory cyclins and phosphorylate hundreds of substrates that control DNA replication, transcription and mitosis. We found that over-expression of PFKFB3 stimulated CDK1 activity in HeLa cells and that purified F2,6BP stimulated recombinant monomeric CDK1 in vitro. We then confirmed the requirement of CDK1 for the pro-proliferative effects of PFKFB3 by demonstrating that CDK1 siRNA but not CDK2, CDK4 or CDK6 siRNA reversed the increased proliferation caused by over-expression of PFKFB3. Importantly, transfection of HeLa cells with PFKFB3-specific siRNA decreased endogenous PFKFB3 which in turn reduced CDK1 activity, increased p27 expression, suppressed G1/S transition, induced apoptosis but had no impact on glucose uptake. These data support a distinct role for PFKFB3 in the regulation of cell cycle progression and apoptosis, and not glucose metabolism. We propose to test the hypothesis that nuclear F2,6BP generated by PFKFB3 activates CDKs and promotes cell cycle progression. We anticipate that nuclear F2,6BP may serve unique roles in regulating CDK1, and other CDKs, and that inhibition of PFKFB3 will suppress CDK activities without significantly affecting glucose metabolism in transformed but not in normal cells.